Preparation process for 20-keto 21(S) hydroxy steroids compounds and intermediates

ABSTRACT

Mixtures of compounds of ##STR1## or a mixture of the latter and a compound of the formula ##STR2## which are useful intermediates for the preparation 20-keto-21(S)-hydroxy steroids.

PRIOR APPLICATION

This application is a division of U.S. patent application Ser. No.071,199 filed Jun. 2, 1993, now U.S. Pat. No. 5,384,419.

OBJECTS OF THE INVENTION

It is the object of the invention to provide an improved process for thepreparation of 20-keto-21(S)-hydroxy steroids and novel intermediatestherefrom.

This and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel process of the invention for the preparation of a compound ofthe formula ##STR3## wherein R₁ is alkyl of 1 to 3 carbon atoms, R₂ isalkyl of 1 to 12 carbon atoms and R₃ is alkyl of 1 to 4 carbon atomscomprises reacting a compound of the Formula ##STR4## wherein R₁, R₂ andR₃ are as defined above in the form of a mixture of isomers on the21-position, with a diastereo selective hydrolysis agent to obtain amixture of the compounds of the Formulas ##STR5## reacting the mixturewith a compound of the Formula

    Hal--SO.sub.2 --R                                          A

wherein Hal is halogen and R is selected from the group consisting ofalkyl of 1 to 6 carbon atoms, --CX₃, and phenyl optionally substitutedwith at least one alkyl of 1 to 3 carbon atoms, and X is fluorine,chlorine, or bromine, to obtain a mixture of a compound of Formula II₁and a compound of the Formula ##STR6## and either reacting the mixturewith an alkali metal acetate to obtain the compound of Formula II₁, andsubjecting the compound of Formula II₁ to solvolysis in a basic mediumor reacting B₃ with a basic and nucleophilic hydroxylating agent toobtain the compound of Formula I.

Examples of R₁ are methyl, ethyl, and propyl, with methyl beingpreferred; examples of R₃ are methyl, ethyl and n-propyl. Examples of R₂are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl,n-hexyl, 2-methyl-pentyl, 2,3-dimethyl-butyl, n-octyl, and2,2-dimethyl-hexyl.

The diastereoselective hydrolysis agent of the mixture of esters ofFormula II can be enzyme such as esterase, or lipase; for example one ofthose known under the names of pancreatic porcine lipase, PS "Amano"(Pseudomonas fluorescens), A₁ "Enzymatix" (animal pancreaticlipase),liposyme 10000 L (a Mucor), N "Amano" (Rhizopus), F₈ "Enzymatix"(lipase), F₃ "Enzymatix" (Mucor javanicus), B₁ "Enzymatix" (PseudomonasSp) or Pseudomonas fluorescens lipase. The lipase known under the namePS "Amano" is preferred. The operation takes place at neutral pH or,preferably, weakly acidic pH, on the order of 5, obtained by the use ofan appropriate aqueous buffer and, if appropriate, the addition of abase. The reaction takes place at ambient temperature or, preferably,slightly higher, on the order of 40° C. It can go forward in thepresence of a solvent which can be an alkane such as hexane or,preferably, a lower alkanol such as n-butanol, propanol, or isopropanol.

The reagent of Formula A is preferably a methane-sulfonyl,ethanesulfonyl, phenysulfonyl, p-toluenesulfonyl ortrifluoromethylsulfonyl chloride or bromide. The reagent is reacted inthe presence of a basic agent which is preferably an amine, such astriethylamine, pyridine, or dimethylamino-pyridine, or a mineral base,such as an alkali metal hydroxide or carbonate in an organic solvent.The solvent is, for example, a halogenated solvent such as methylenechloride, chloroform, or dichloroethane or an aromatic or saturatedhydrocarbon, such as benzene, toluene, or cyclohexane.

The inversion of the sulfonate of Formula IV in a mixture with thecompound of Formula II₁ can be carried out with an SN₂ -typenucleophilic substitution, by using an alkali metal acetate, especiallysodium acetate or potassium acetate. The operation is carried out byheating in an aprotic polar organic solvent such as bis(2-methoxy ethyl)ether, dimethylformamide, dimethylacetamide, dimethylsulfoxide, ordimethylpyrrolidone.

The solvolysis is preferably an alcoholysis carried out in the presenceof sodium hydroxide or potassium hydroxide. The alcohol used is a loweralkanol, preferably methanol or ethanol. The solvolysis can also be ahydrolysis using for example the bases mentioned above.

The inversion of the sulfonate can also be carried out by the action ofa basic and nucleophilic hydroxylating agent which can be alkali metalhydroxide or alkaline-earth metal hydroxide, e.g. sodium hydroxide,potassium hydroxide, calcium hydroxide, or barium hydroxide. Theoperation is carried out in a solvent such as mixtures oftetrahydrofuran, bis(2-methoxyethyl) ether, triethyleneglycol dimethylether, dimethylformamide, dimethylsulfoxide, dimethylacetamide withwater.

A quite particular subject of the invention is the process wherein thecompound of Formula II has R₁, R₂ and R₃ all being methyl. Finally, theinvention includes, as new industrial compounds, the mixture of thecompounds of Formulas II₁ and III and the mixture of the compounds ofFormulas II₁ and IV as defined previously.

The compounds of Formula I are endowed with progestomimetic andanti-estrogen activity, and are described in European Patent No. 7823.The starting compounds of Formula II are described in this same Patent.

The different enzymes mentioned above are commercially available productfrom, for example, the following companies: Amano Pharmaceutical Co.Ltd, Enzymatix Ltd, Sigma Chemie SARL, and Novo Nordisk Bio Industrie.

In the following examples, there are described several preferredembodiments to illustrate the invention. However, it should beunderstood that the invention is not intended to be limited to thespecific embodiments.

Example 1: 17α-methyl-17β-(2S-hydroxy-1-oxo-propyl)-Δ4,9-estradien-3-one

Step A: Mixture of17α-methyl-17β-(2R-hydroxy-1-oxo-propyl)-Δ4,9-estradien-3-one and17α-methyl-17β-(2S-acetoxy-1-oxo-propyl)-Δ-estradien-3-one.

5 g of 17α-methyl-17β-(2R, S-acetoxy-1-oxo-propyl)-Δ4,9-estradien-3-one,5 g of PS "Amano" lipsase, 7 ml of n-butanol, and 18 ml of pH=5 bufferwere mixed together and the temperature was maintained at 37° C. whilethe pH was kept at 5 by the addition of 1N sodium hydroxide. After 144hours, 3.75 ml of pH=5 buffer, 1.25 g of PS "Amano" lipase were addedand, after 168 hours, extraction took place with methylene chloride. Theorganic phase was washed with water, dried, and the solvent wasevaporated to obtain 4.63 of the expected mixture containingapproximately 50% of each of the constituents.

NMR Spectrum CDCl₃ (300 MHz)

--50/50 mixture of 2 constituents

1) 0.81 (s): 18-CH₃ ; 1.16 (s): 17-CH₃ ; 1.33 (d) CH₃ --CH 3.71 (d):OH--; 4.56 (quintuplet): --CH--OH; 5.68 (s) : H₄

2) 0.82 (s): 18-CH₃ ; 1.21 (s): 17-CH₃ ; 1.38 (d): CH₃ --CH--; 2.11 (s):O--Ac; 5.42 (q) : CH₃ --CH--; 5.68 (s) : H₄.

The respective proportions of the two constituents of the mixture werealso determined by HPLC comparatively with the pure constituentsdescribed and characterized in European Patent No. 7823 (Example 2).

Step A': Mixture of17α-methyl-17β-(2R-hydroxy-1-oxo-propyl)-Δ4,9-estradien-3-one and17α-methyl-17β-(2S-acetoxy-1-oxo-propyl)-Δ4,9-estradien-3-one.

0.1 g of17α-methyl-17Δ-(2R,S-acetoxy-1-oxo-propyl)-Δ4,9-estradien-3-one, 2 ml ofphosphate PH 7 buffer, and 20 mg of the enzyme shown in the table belowwere mixed together; the mixture was stirred (300/revs/min) while the pHwas adjusted to maintain it between 5 and 8. The expected mixture wasobtained after stirring 64 to 137 hours with (R) ester being hydrolyzedin the proportions shown in the table.

The reaction was followed by thin layer chromatography eluting with atoluene-ethyl acetate mixture (1-1). The composition of the finalmixture was determined by HPLC on a PARTISIL No. 2102 D column, elutingwith a hexane-methylene chloride-dioxane mixture (8-1-1). The followingresults were obtained:

    ______________________________________                                                         % of R and alcohols                                          Enzyme use       in the medium                                                ______________________________________                                        PS Amano lipase  >99.5% of (R)                                                A.sub.1 enzymatix lipase                                                                       85% (R) - 15% (S)                                            10 000 L liposyme                                                                              90% (R) - 10% (S)                                            B.sub.1 enzymatix lipase                                                                       >95% of (R)                                                  Pseudomonas fluorescens                                                                        >90% of (R)                                                  ______________________________________                                    

Step B: Mixture of17α-methyl-17β-(2R-methyl-sulfonyloxy-1-oxopropyl)-Δ4,9-estradien-3-oneand 17α-methyl-17β-(2S-acetoxy-1-oxopropyl)-Δ4,9-estradien-3-one.

2 g of the product of Step A and 10 ml of methylene chloride were mixedtogether under an inert gas atmosphere and then 0.8 ml of triethylaminewere added to the solution. The mixture was cooled to 0° C./-2° C. andthen 0.35 ml of methane sulfonyl chloride were slowly added whilemaintaining the temperature of 0° C. to 2° C. The mixture was stirredfor 1 hour and was then poured into 5 ml of water. Extraction wascarried out with methylene chloride and the organic phase was washedwith water, dried, and the solvent was evaporated to obtain 2.35 g ofthe expected mixture composed of approximately 46% of the (S) acetateand 54% of the (R) mesylate. (Determination by HPLC on PARTISIL columnusing hexane-methylene chloride-dioxane 80/10/10).

NMR Spectrum CDCl₃ (300 MHz)

50/50 mixture of the 2 constituents

1) 0.90 (s): 18-CH₃ ; 1.56 (d): CH₃ --CH--; 3.16 (s): O--SO₂ --CH₃.

2) 0.82 (s): 18-CH₃ ; 1.39 (d): 17-CH₃ --CH--; 2.11 (s): OAc; and, inaddition, 1.18 (s) and 1.20 (s): 17-CH₃ ; 5.42 (q) and 5.52 (q):--OC--CH--CH₃ ; 5.68 (s): H₄

Step C: 17α-methyl-17β-(2S-acetoxy-1-oxo-propyl)-Δ4,9-estradien-3-one

1 g of the product of Step B and 7 ml of bis (2-methoxy ethyl) eitherwere mixed together under an inert gas atmosphere and 0.38 g ofpotassium acetate were added to the solution followed by heating to 90°C. for 22 hours. After cooling to ambient temperature, the mixture waspoured into 20 ml of water, and stirred for 4 hours. The,product wasseparated, rinsed with water and then dried to obtain 0.77 g of theexpected product containing approximately 94% of isomer (S).

(Determination by HPLC as in Step B.)

NMR Spectrum CDCl₃ (300 MHz)

Presence of approximately 95% of the constituent characterized asfollows:

0.82 (s): 18-CH₃ ; 1.20 (s).17-CH₃ ; 1.39 (d) and 5.42 (q): CH₃ --CH--;2.11 (s): O--Ac; 5.69 (s): H₄.

Step D: 17α-methyl-17β-(2S-hydroxy-1-oxopropyl)-Δ4,9-estradiene-3-one

0.5 g of the product of Step C, 4 ml of methanol and 0.5 ml ofmethanolic potassium hydroxide at 0.06 g/5 ml were mixed together andthe solution was stirred for 4 hours at 20° C., then neutralized by theaddition of 0.7 ml of 0.1N hydrochloric acid. 5 ml of methylene chlorideand 3 ml of water were added, followed by decanting and reextracting theaqueous phase with methylene chloride. The combined organic phases weredried and the solvent was evaporated to obtain 0.427 g of the expectedproduct containing 94% of (S) isomer.

(Determination by HPLC as in Steps B and C).

NMR Spectrum CDCl₃ (300 MHz)

Presence of approximately 95% of 21(S) compound as characterized inExample 1 of European Patent No. 7823 (isomer A).

Example 2: 17α-methyl-17β-(2S-hydroxy-1-oxo-propyl)-Δ4,9-estradien-3-one

100 mg of the product of Step B of Example 1, 3 ml of solvent, and 100mg of base were mixed together under an inert gas atmosphere. Themixture was heated with stirring and the reaction was monitored by HPLC.(Partisil column-eluant: hexane-methylene chloride-dioxane 8/1/1) Theprecise conditions and the results are shown in the table below.

    ______________________________________                                                                    Duration                                                                             % of expected                                                 Tempera- of     products in the                            Base   Solvent     ture     reaction                                                                             crude product                              ______________________________________                                        Ca(OH).sub.2                                                                         Diglyme* +  90° C.                                                                           1 H   80%                                               water                                                                  NaOH   Dimethyl    80° C.                                                                          10 H   73%                                               acetamide +                                                                   water                                                                  Ba(OH).sub.2                                                                         Tetrahydro- 80° C.                                                                          24 H   67%                                               furan + water                                                          ______________________________________                                         *bis(2-methoxyethyl) ether                                               

Various modifications of the process of the invention may be madewithout departing from the spirit or scope thereof and it is to beunderstood that the invention is intended to be limited only as definedin the appended claims.

What we claim is:
 1. A mixture consisting of compounds of ##STR7## or amixture consisting of the compound of Formula II₁ and a compound ofFormula ##STR8## wherein R₁ is alkyl of 1 to 3 carbon atoms, R₂ is alkylof 1 to 12 carbon atoms, R₃ is alkyl of 1 to 4 carbon atoms and R isselected from the group consisting of alkyl of 1 to 6 carbon atoms,--CX₃ and phenyl optionally substituted with at least one alkyl of 1 to3 carbon atoms and X is fluorine, chlorine or bromine.